1. Field of the Invention
The present invention relates to the field of enzymatic engineering, and more particularly relates to a novel leech hyaluronidase.
2. Description of the Related Art
Hyaluronic acid (HA) is a linear and unbranched high-molecular-weight polysaccharide composed of repeating disaccharide D-glucuronic acid (GluUA) and N-acetyl-D-glucosamine (GlcNAc) units linked through β-1,4 bonds. High-molecular-weight HA is widely distributed among various host tissues and participates in numerous physiological processes. The biological functions and applications of HA depend on its molecular mass. In particular, low-molecular-weight HA oligosaccharides have unique biological activities. Smaller HA oligosaccharides can stimulate fibroblast proliferation and collagen synthesis and selectively kill many types of cancer cells via disruption of the receptor-hyaluronan interaction. In addition, low-molecular-weight HA oligosaccharides are easily absorbed by the body and serve as precursors for the synthesis of both higher-molecular-weight HA molecules and other substances. Thus, a specific narrow spectrum of HA oligosaccharides could have broad applications in medicine, food and cosmetics. Low-molecular-weight HA is mainly produced by the degradation of high-molecular-weight HA by physical and chemical methods. However, the products of these methods have a broad range of molecular weight, making it difficult to obtain HA oligosaccharides with specific molecular weight. Many chemical approaches are time-consuming. Rare carbohydrate oligosaccharide backbones and expensive substrate also limited the large scale application of those chemical methods. In contrast, the enzymatic production of HA oligosaccharides with a well-characterized HAase is promising and attractive because of its unique advantages, such as mild operation conditions and high product specificity.
Hyaluronidases (HAases) which can degrade HA are found to be involved in many important biological processes, such as cell division, cell connection, activity of germ cell, DNA transfection, embryonic development, tissue repair and cell proliferation. HAases are a large family of glycosidase that are widely distributed in eukaryotes and procakyotes. According to substrate specificities and hydrolysis products, HAases are divided into three classes: hyaluronate 4-glycanohydrolases (EC 3.2.1.35, Bovine testicular hyaluronidase, BTH), hyaluronate lyases (EC 4.2.2.1, Streptococcus hyaluronate lyase) and hyalutonate 3-glycanohydrolases (EC 3.2.1.36, Leech HAases).
Hyaluroniases from leech is a representative enzyme of the third class of hyaluroniases. Leech HAase has higher substrate specificity and a narrow-spectrum. It degrades high-molecular-weight HA to HA tetrasaccharides (HA4) by catalyzing the hydrolysis of β-1,3-glucosidic bond. Because of its high substrate specificity, leech HAase can not degrade chondroitin or chondroitin sulfate. In addition, activity of leech HAase is unaffected by heparin. Therefore, leech HAases have great potential in clinical applications.
Currently, leech HAase is mainly obtained by extracting from living leech tissue. The limited source and tedious extraction process have impeded the application of leech HAase in medical application and scientific research.
There is a need for providing an easy source of leech HAases and an effective method of producing low-molecular-weight HA using the HAases. The present invention sacrifices this need and provides other benefits as well.